The genus Amphitetranychus Oudemans (Tetranychidae) consists of only three species, A. quercivorus (Ehara & Gotoh), A. savenkoae (Reck) and A. viennensis (Zacher). The original description of A. savenkoae was extremely simple and had no drawing of the aedeagus; however, a subsequent study described only the aedeagus. The present study investigated all three species in detail using a combination of morphological traits, crossbreeding experiments, esterase zymograms and the mitochondrial cytochrome c oxidase subunit I (COI) gene. Morphological differences in the peritremes and male aedeagi were observed among the three species. Complete reproductive isolation was confirmed in the reciprocal crosses between the morphologically similar A. savenkoae and A. quercivorus (no female offspring were produced). Esterase zymograms differed interspecifically, but not intraspecifically (among individuals in a given species). All three species formed clearly separate clades with 100% bootstrap values in the COI tree, and A. savenkoae was more closely related to A. quercivorus than to A. viennensis, which corresponded to the morphological similarity of their aedeagi and setal counts on tarsi IV. A key to Amphitetranychus species is provided.